Toxoplasma gondii and Rickettsia spp. in ticks collected from migratory birds in the Republic of Korea

Migratory birds disperse ticks and associated tick-borne pathogens along their migratory routes. Four selected pathogens of medical importance (Coxiella burnetii, Rickettsia spp., Francisella tularensis, and Toxoplasma gondii) were targeted for detection in 804 ticks (365 pools) collected from migratory birds at Hong and Heuksan Islands in the Republic of Korea (ROK) from 2010 to 2011 and 2016. Toxoplasma gondii and Rickettsia spp., were detected in 1/365 (0.27%) and 34/365 (9.32%) pools of ticks, respectively. T. gondii and five rickettsial species were recorded in ticks collected from migratory birds for the first time in ROK. The five rickettsial species (R. monacensis, Candidatus Rickettsia longicornii, R. japonica, R. raoultii, and R. tamurae) were identified using sequence and phylogenetic analysis using ompA and gltA gene fragments. Rickettsia spp. are important pathogens that cause rickettsiosis in humans, with cases recorded in the ROK. These results provide important evidence for the potential role of migratory birds in the introduction and dispersal of T. gondii and Rickettsia spp. along their migratory routes and raise awareness of potential transmission of zoonotic tick-borne pathogens associated with migratory birds in the ROK.

dispersal of Severe Fever with Thrombocytopenia Syndrome (SFTS) virus present in China, Japan, and Korea 19 . Consequently, identification of tick species and associated tick-borne pathogens and host migratory birds are important to assess the potential risk of tick-borne disease introductions in each region where there are suitable habitats for migratory birds.
In the ROK, Heuksan-do (do = island), Hong-do, and Nan-do are stopover habitats of migratory birds that are located in the Yellow Sea. Ticks collected from migratory birds were identified to species, that included eight species (Haemaphysalis flava, H. formosensis, H. longicornis, H. concinna, H. ornithophila, Ixodes nipponensis, I. turdus, and Amblyomma testudinarium) belonging to three genera. Of the eight species, I. turdus and H. flava were the most prevalent species collected [20][21][22] . Only three tick-borne microorganisms (Borrelia spp., A. phagocytophilum, and Bartonella grahamii) were detected in these ticks. Borrelia spp. were the most prevalent microorganisms detected in I. turdus and H. flava, while A. phagocytophilum and B. grahamii were detected only in I. nipponensis and I. turdus, respectively 21,22 . However, information for other tick-borne microorganisms such as Coxiella burnetii, Rickettsia spp., Toxoplasma gondii, and Francisella tularensis in ticks collected from migratory birds remain unknown. Infections of these pathogens in humans in the ROK were recorded and potentially influence public health 23-28 . Accordingly, this study aimed to extend our previous work 22 to survey for the presence of four tick-borne pathogens, C. burnetii, T. gondii, F. tularensis, and Rickettsia spp., in ticks collected from migratory birds at two islands, Hong-do and Heuksan-do, ROK. Sequencing and phylogenetic analysis were done for species identification of Rickettsia spp.

Results
Tick-borne microorganisms in bird ticks. A total of 804 ticks belonging to three genera and seven species were placed in 365 pools according to bird host, date and location of collection, and stage of development ( Sequencing and phylogenetic analysis. Toxoplasma gondii was confirmed by sequence analysis of repetitive DNA fragments from nested conventional PCR (504 bp). Comparison of generated sequences (Suppl .  Table S1) with deposited sequences on NCBI databank showed 100% identity with T. gondii sequences detected from mice in India (NCBI accession No.: KC607824) and cattle and goats in Iraq (NCBI accession No.: KX963353 and KX963355).
Detection of Rickettsia spp. targeting ompA and gltA gene fragments from 34 Rickettsia spp. positive tick pools showed that 30 and 34 pools were positive, respectively. The sequences of ompA and gltA genes were deposited on NCBI with an accession number of each sequence as shown in Table 2 and Suppl. Tables S2 and  S3. Variations among the sequences of ompA and gltA gene fragments were observed. The percent sequence identity among sequences of ompA and gltA was 78.7% and 93.7%, respectively. Sequences of the gltA gene were divided into five Rickettsia spp. groups, while the ompA gene was separated into four Rickettsia spp. The percent identity among the generated sequences for each group ranged from 97.2 to 100.0%. Comparison of generated sequences of the ompA and gltA gene fragments to the deposited sequences of Rickettsia species on NCBI and phylogenetic analysis showed that the detected Rickettsia spp. belong to five species (R. monacensis, Candidatus Rickettsia longicornii, R. japonica, R. raoultii, and R. tamurae) with the sequence similarity ranging from 98.80 to 100.00%, while 3 specimens could not be identified to species ( Fig. 1; Table 2). Although phylogenetic analysis of ompA gene showed that the detected strains (HS40, HS46, HS63, HS76, HS81, HS129, H78, and H179) were in the same clade with Ca. R. longicornii and Ca. R. jingxinensis (Fig. 1), the sequence analysis showed a higher similarity (100%) of detected strains to Ca. R. longicornii than Ca. R. jingxinensis (99.2%) (Suppl . Table S2). Therefore, the detected strains were identified as Ca. R. longicornii. Based

Discussion
Rickettsia spp. and T. gondii were detected in pools of ticks collected from migratory birds. The results provide additional information about microorganisms harbored by ticks infesting migratory birds in the ROK. A total of five tick-borne microorganisms, including Borrelia spp., A. phagocytophilum, B. grahamii, T. gondii, and Rickettsia spp., have been recorded in ticks collected from migratory birds in the ROK 21,22 . Rickettsia spp. and Borrelia spp. were the most prevalent tick-borne microorganisms detected from Ixodes spp., and the results are consistent with previous reports from other countries [12][13][14][15][16]18 . However, there was a high infection rate and greater species diversity of Rickettsia species observed among ticks collected from migratory birds in the ROK compared to reports from other countries. Human infections of R. monacensis, R. japonica, and R. raoultii have been documented in the ROK [27][28][29] , and R. tamurae in Japan 30 . Therefore, ticks from migratory birds likely play a certain role in the transportation of ticks and associated rickettsial pathogens to these islands and the Korean mainland. Toxoplasma gondii has been detected in birds in other areas of the world 31,32 , and the potential role of ticks and migratory birds in dispersing T. gondii was suggested 33,34 . However, no evidence of T. gondii carried by bird ticks had been previously provided. In this study, one female I. turdus tick collected from a pale thrush was positive for T. gondii, this is the first report of T. gondii detected in I. turdus in the ROK. Therefore, the T. pallidus and associated ticks may have contributed to the spread of T. gondii along its migratory routes. Further studies on the presence of T. gondii in T. pallidus bird and direct transmission of T. gondii by I. turdus need to be conducted.    [20][21][22] . However, infections of these pathogens in related bird species has not been characterized in the ROK. There are stopover habitats in the ROK for migratory birds on their migration routes between the northeastern Palearctic region, including Russia and eastern China, and southeast Asia 21 . The presence of tick-borne pathogens (B. garinii, A. phagocytophilum, and E. chaffeensis) in migratory birds was confirmed in China 35 . These results suggest that the migratory birds collected in the ROK may be infected and become important natural reservoirs of these tick-borne pathogens. Therefore, it is necessary to conduct further studies on the surveillance of tickborne pathogens in migratory and resident birds and local tick and animal/bird reservoirs to better understand the role of migratory birds in the potential introduction and spread of tick-borne pathogens.
Wild birds are known to be reservoir hosts of C. burnetii and F. tularensis and associated ticks might transmit the pathogens to human 36,37 . Ixodes ricinus infesting birds were suggested to be the vectors of C. burnetii 37,38 . In the ROK, C. burnetii and F. tularensis were detected more frequently in H. longicornis and H. flava ticks collected from the environmental habitats and domestic or wild animals [39][40][41] . However, the two pathogens were not detected in ticks infesting wild birds in this study, and the presence of these two pathogens in ticks feeding on birds in China and other southeast Asian countries located along their migration routes 21 has not been recorded.
Surveillance of C. burnetii, F. tularensis, Rickettsia spp., and T. gondii in this study demonstrated the presence of T. gondii in ticks collected from migratory birds. Rickettsia spp., including R. monacensis, Ca. R. longicornii, R. japonica, R. raoultii, and R. tamurae, were the most commonly detected microorganisms in ticks collected Table 2. The identified species of Rickettsia in bird ticks collected from 2010 to 2011 and in 2016. *Only gltA gene was amplified. **These species may be also regarded as residents or partial migrants in Korea, but the birds in this study were all true migrants that were crossing the national borders and ecological barriers like the Yellow Sea.  These two islands are located in the southwestern tip of the Korean Peninsula, most birds captured in this study were true migrants that were crossing a national border and an ecological barrier, the Yellow Sea. Tick collected from 2010 to 2011, pooled by species and stage of development, were designated as H1-H195 and in 2016 they were designated as HS1-HS184. Samples in this study were shared with those in the analysis of Anaplasma and Borrelia species in a previous study 22 , while a few ticks were supplemented to replace destroyed samples for the analysis. Detailed information on collection sites, bird collections, and tick collections were reported in Seo et al. 22 . Ticks were identified using standard morphological keys [43][44][45] , and then placed in pools according to collection location and date, stage of development, sex, and, host species. Nymphs and larvae were placed in pools of 1-6 and 1-9 ticks, by species and stage of development, respectively, while adult ticks were assayed individually 22 . The pooled samples were placed in 1.5 ml cryovials containing 70% ethanol and stored at -80 °C until analysis.
DNA extraction. After washing three times using UltraPure™ DNase/RNase-Free distilled water (Thermo Fisher Scientific, USA), the tick samples were placed in a tissue grinding tube (SNC, Hanam, Korea) containing 0.6 mL phosphate-buffered saline and 2.3 mm stainless-steel beads and then homogenized using Precellys 24 Tissue Homogeniser (Bertin Instruments, Montigny-le-Bretonneux, France). The homogenate was centrifuged at 300×g for 1 min and the supernatant was collected for total nucleic acid extraction using Maxwell ® RSC Viral Total Nucleic Acid Purification Kits (Promega, USA) and an automated Maxwell RSC Instrument (Promega). PCR analysis. Primers and PCR conditions for detection of the selected four targets are shown in Table 3.
DNA used for positive control in PCR detection of C. burnetii from Nine Mile strain, and of T. gondii was from the strain G-P-14-7 that was isolated and stored in Animal and Plant Quarantine Agency, South Korea. Positive control DNA of F. tularensis and Rickettsia spp. was chemically synthesized according to the sequence information on NCBI with accession No. was CP073128 (F. tularensis) and CP047359 (R. japonica). Recombinant DNA carrying standard fragments were constructed using the pGEM ® -T vector system (Promega, Madison, WI, USA) and PCR products amplified by each detection primer pair. Detection of C. burnetii was done by two successive PCRs, conventional PCR was performed using primer pair Trans1/2 (Table 3), followed by nested real-time PCR (qPCR) using primer pair Cox111-F/R (   48 . Identical sequences of Rickettsia acquired from the NCBI were used for alignment together with generated sequences using Clustal X version 2.0 49 , and Maximum likelihood phylogenetic trees were created using the Kimura 2-parameter model that estimate evolutionary distance based on the nucleotide substitutions 50 , gamma distribution, and bootstrapping 1000 times with MEGA7 software 51 . Ethics approval. All field procedures including bird capture, handling, and sampling were under the bird banding station licenses (#501000085200500002, 2011-8, 2016-1, and 2016-2) issued by the local government (Shinan Country), the Korean Ministry of Environment (Yeongsan River Environmental Office), and the Cultural Heritage Administration. This study was approved by The Korea National Park Service (KNPS). Captured birds were safely and ethically examined, sampled, and released safely following the institutional guideline (National Park Research Institute, KNPS) for constant-effort bird banding surveys in Korean National Parks 42 .

Data availability
All data generated or analysed during this study are included in this published article (and its Supplementary file). Generated sequences were deposited on NCBI with accession number OL687165-OL687228.